Ascorbic acid, which is also called vitamin C, is an important vitamin for its shortage in vivo causes scorbutus, etc., and the relation between ascorbic acid and senility has been noted in recent years. Therefore, the determination of ascorbic acid is recognized as important.
Previous methods for the quantitative determination of ascorbic acid include chemical methods such as the hydrazine method and instrumental methods such as high performance liquid chromatography. However, these known methods have the disadvantages of involving complicated procedures and requiring many hours for the determination.
Recently, ascorbate oxidase (hereinafter referred to as ASOD) was discovered which catalyzes oxidation reaction of reduced ascorbic acid in the presence of oxygen to form oxidized ascorbic acid and hydrogen peroxide. Ascorbic acid in a sample is determined by measuring the amount of oxygen consumed or the amount of hydrogen peroxide formed by this enzyme reaction [JP-A-209770/94, JP-A-23971/96 (EP-A-682116)].
JP-A-209770/94 discloses a chemical method as the specific method for the determination of hydrogen peroxide, but does not disclose a method using peroxidase. JP-A-23971/96 (EP-A-682116) discloses a method for the determination of hydrogen peroxide which comprises reacting hydrogen peroxide, in the presence of peroxidase, with a chromogen comprising a hydrogen donor such as phenol or a Trinder's reagent and a coupler such as 4-aminoantipyrine to form a pigment, and then measuring the absorbance of the reaction solution colored by the formed pigment. This is a method for the determination of hydrogen peroxide formed which is carried out after the reaction for forming hydrogen peroxide is completed and reduced ascorbic acid is completely converted into oxidized ascorbic acid.
Generally, it is known that the presence of reducing substances such as ascorbic acid and dithiothreitol disturbs a reaction for forming a pigment by the use of peroxidase. Therefore, it is necessary to carry out the reaction for forming a pigment only after reduced ascorbic acid in a sample is completely oxidized by the action of ASOD and the formation of hydrogen peroxide is completed. In this method, despite of the utilization of an enzyme reaction, chemically unstable hydrogen peroxide must be accumulated once in the reaction solution.
Biological samples contain reducing substances such as uric acid, catalase, bilirubin and hemoglobin which consume the formed hydrogen peroxide. Therefore, the above method in which hydrogen peroxide is accumulated can not give accurate data having reproducibility. Further, the method involves complicated procedures and requires many hours for the determination.
In a living organism, oxidized ascorbic acid exists along with reduced ascorbic acid, and the determination of total ascorbic acid composed of reduced ascorbic acid and oxidized ascorbic acid is also clinically important.
Total ascorbic acid can be determined by converting oxidized ascorbic acid into reduced ascorbic acid with a reducing agent such as dithiothreitol and measuring reduced ascorbic acid using ASOD. However, the method in which hydrogen peroxide formed is reacted with a chromogen in the presence of peroxidase and the formed pigment is determined has the disadvantage that the formation of the pigment is disturbed because the chromogen is influenced by a coexisting reducing agent. To solve this problem, elimination of the remaining reducing agent from the reaction solution prior to the determination of hydrogen peroxide becomes necessary, which makes the procedures complicated.
JP-A-29297/82 (U.S. Pat. No. 4,384,042) and JP-A-218069/85 disclose methods for the determination of an analyte in a sample wherein the reaction of the sample with oxidase acting on the analyte as the substrate to form hydrogen peroxide (hereinafter referred to as the hydrogen peroxide-forming reaction) and the reaction of the hydrogen peroxide with a chromogen to form a pigment (hereinafter referred to as the pigment-forming reaction) are carried out in the same reaction system. However, in these publications, there is no disclosure of a method wherein the oxidation of reduced ascorbic acid with ASOD and the reaction of the formed hydrogen peroxide with a chromogen to form a pigment are carried out in the same reaction system.
As previously described, the formation of a pigment is inhibited in the presence of a reducing substance such as ascorbic acid, and thus it was considered impossible to carry out the hydrogen peroxide-forming reaction and the pigment-forming reaction in the same reaction system. However, it has been found that there exist chromogens which are not affected by a reducing substance under specific conditions in the determination process, and the present invention has been completed based on this finding.